SSR MINING IN 454 TRANSCRIPTOME SEQUENCING-DERIVED MUSA ACUMINATE UNIGENES

Massively parallel pyrosequencing-based transcriptome analysis is an efficient approach for large-scale, functionally relevant, gene-derived SSR discovery. The objectives of this study were to identify SSR loci in unigene sequences generated using 454 transcriptome pyrosequencing, and to verify predicted gene function and in silico-derived differential expression. cDNA libraries were prepared from total RNA material extracted from leaves of ‘Calcutta-4’ (Musa acuminate ssp. burmannicoides) and ‘Grande Naine’ (AAA, Cavendish), both uninoculated and inoculated with Mycosphaerella musicola (the causal agent of Sigatoka leaf spot). ‘Calcutta-4’ is a wild diploid widely used as a donor of biotic stress-resistance genes in conventional breeding programs for improved polyploids. cDNA libraries for each challenged genotype were submitted to 454 sequencing, with 24,246 and 23,729 contigs identified for ‘Calcutta-4’ and ‘Grande Naine’, respectively. In silico-based gene function prediction was made against the NCBI Genbank database. Contigs were analyzed for SSRs using the program mreps and flanking primer pairs designed using Primer3. A total of 4098 and 4096 SSR loci, where flanking primers could be designed, were identified in ‘Calcutta-4’ and ‘Grande Naine’ datasets, respectively. From these, 75% are associated with unigenes-coding proteins with known function, 0.13% with unigenes potentially involved in defense or stress responses, and 96% associated with genes differentially expressed under conditions of infection and non-infection by M. musicola. Overall, microsatellites were present in 24% of unigenes with predicted function. Marker validation in M. acuminate diploid material contrasting in resistance to black leaf streak and Sigatoka leaf spot is currently ongoing. The microsatellites identified, particularly those associated with genes potentially involved in defense or stress responses, will serve as useful tools for employment in Musa genetic improvement programs.